Lessons Learned from Pharmaceutical Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 118

6.3.2.2 Immunotoxicity
Complex processes such as immune response are not simple to analyze. The different
types of nanopharmaceuticals and their proposed route of application can activate
various immune defense mechanisms. In the past, apart from vaccine delivery, only
little attention was paid to the immunostimulatory effects of nanopharmaceuticals.
One opportunity to detect an immunological effect of nanopharmaceuticals is to
investigate the complement system after treatment. The complement system consists
of several proteins, both in serum and on cell surfaces and after activation it is capa-
ble of opsonization, chemotaxis, and cell lysis. The opportunity to measure compo-
nents of the complement system is possible (Nilsson and Ekdahl 2012). Cytokine
release and dendritic cell activation are suitable tests to evaluate the potential of
nanopharmaceuticals to impact leukocyte recruitment and inflammation. A further
strategy is to determine the antibody response. There are some assays but these are
not well established yet (Jung et al. 2001). The detection of bioaccumulation and its
potential also needs more attention. Although biodegradability and biocompatibility
are designed from the outside, it has to be proven that the nanoparticulate formula-
tions stay only as long as necessary and wanted in the human body.
6.3.2.3 Genotoxicity or Mutagenicity
Some materials are able to induce genetic mutations. The Organization for Economic
Co-operation and Development (OECD) validated various in vitro methods for
genetic toxicology testing.
Single cell gel electrophoresis also called Comet assay is a technique to analyze
DNA damage in individual cells. It is sensitive to damage above 50 breaks to a maxi-
mum of 10,000 (Olive and Banath 2006). In the field of nanopharmaceuticals, the
Comet assay is the most applied one until now. The Bacterial Reverse Mutation Assay
also referred to as Ames test is a fast method to detect mutagenic properties of materi-
als. Bacteria with a defect DNA repair mechanism are not able to recover DNA dam-
age after treatment with mutagen substances, which leads to an increase in the number
of revertant colonies relative to the background level. The GreenScreen HC genotoxic-
ity assay is a yeast DNA repair reporter assay with the possibility for high throughput
(Cahill et al. 2004). No single mutagenicity test so far is validated enough (in particular
for nanoparticles) and provides positive controls for mutagenic nanoparticles to act as
a standalone technique. The OECD recommends several other methods for determi-
nation of damages in genetic material in their “Guidelines for Testing of Chemicals.”
Nanoparticle formulations could possess the capacity to act as a teratogen, which
leads to the investigation of their teratogenicity. An animal model (zebra fish) was
described to clarify the potential of formulations in relation to physiological mal-
development (Brannen et al. 2010). However, it is important to mention that inves-
tigations based on animal testing should only be performed in very late stages of
development and may be surrogated by alternative methods using embryonic stem
cells (Scholz et al. 1999; Rohwedel et al. 2001; McNeish 2004).
6.3.2.4 Alterations in Cell Function
Alterations in cell function might also occur, for example, as a result of changes
in surface proteins or shifts in gene expression patterns (as can occur via miRNA